Microarrays are powerful tools for comprehensive analysis of biomolecule interactions, including protein-protein and oligonucleotide-oligonucleotide interactions. Such analysis is useful in molecular characterization and diagnosis of physiological or disease states and has a broad potential. In all microarrays, interactions are analyzed by first immobilizing a set of biomolecules in an array format on a slide. The slide is then probed with a set of fluorescently-labeled complementary ligands and any binding is noted.
Compared to the DNA microarrays, the fabrication of useful protein microarrays is generally more difficult and technically challenging. This is because, proteins are intrinsically fragile molecules which are sensitive to exposure to both low and high temperature, extremes of pH, presence of hydrophobic surfaces, high shear and to removal of water. It is imperative therefore that such conditions are avoided during preparation, storage and handling of protein microarrays.
A preferred solution to many of these problems is to attach proteins to encoded microbead particles, including encoded particles made of polymer resin (“Multianalyte Molecular Analysis Using Application-Specific Random Particle Arrays,” U.S. application Ser. No. 10/204,799, filed on Aug. 23, 2002; WO 01/98765, incorporated by reference). The encoded capture-protein coated particles are then assembled in a 2D array format and placed in contact with samples anticipated to contain target proteins. Any binding between the capture and target proteins are then determined by the presence of a fluorescent assay signal. Particular capture proteins generating a positive assay signal can be determined by decoding the array. There are several known and commercially available methods for immobilization of proteins on microparticles (Bangs Laboratories Inc., TechNote # 205 Covalent Coupling, 2002 and TechNote # 204, Adsorption to Microspheres, 1999). Most commonly used approaches result in random covalent attachment or sticking of proteins onto microparticle surfaces, which often leads to a wrongly oriented molecule incapable of participating in binding. In addition, use of improper chemistry per se may chemically modify and hence denature the protein molecule.
Coupling proteins to surfaces using site-selective chemistry can circumvent some of these problems. In principle, such oriented attachment leaves the proteins' active sites accessible and also improves their stability (Peluso, P. et al., Analytical Biochemistry 312 (2003) 113-124). Such techniques are, however, of restricted use because they require additional protein modification, purification and concentration steps, which may be impractical for use with large numbers of unique molecules. Hence the choice of a surface chemistry and surface topology that will allow diverse types of proteins to be immobilized and yet retain their secondary structure and thus their biological activity is needed.
Hydrogels are three-dimensional hydrophilic polynmeric networks capable of imbibing large quantities of water. Their high aqueous content offers a “protein-friendly” environment and they have recently received attention for their potential use as a microarray substrate
(see Perkin Elmer Corp.'s website: HydroGel Application Note). Arenkov et al. (Arenkov, P. et al. Analytical Biochemistry, 278, 123-131 (2000)) reported the fabrication of arrays which were produced by immobilizing proteins in gel pads (100 μm×100 μm×20 μm) which were in turn attached to a glass slide surface. Because of three-dimensional matrix structure, the protein immobilization was reported to be very efficient. The aqueous environment helped to keep the protein in its native form and it was freely accessible for assay binding reactions. The major disadvantages of the method are a complicated fabrication process and the difficulty of removing the unbound protein from the gel pad, due to transport limitations.
None of these approaches, therefore, are sufficiently versatile to provide a broad platform for multiplexed protein-ligand interaction analysis which is compatible with the vast diversity of protein structures and functions and permits maintenance of secondary and tertiary protein structure.